Oplophorus gracilirostris luciferase from the deep-sea shrimp is a secretory-type luciferases and uses coelenterazine as a luminescence substrate (Patent Document 1, Non-Patent Document 1). Oplophorus luciferase consists of a 35 kDa protein and a 19 kDa protein with the molecular weight of a 106 kDa protein (Non-Patent Document 2). In Oplophorus luciferase, the 19 kDa protein is found to be a catalytic domain of the luminescence reaction (Non-Patent Document 2). By comparison of Oplophorus luciferase with other marine luciferases, it is known that the substrate specificity of Oplophorus luciferase is broad and coelenterazine analogues are used as good luminescence substrates (Non-Patent Document 3). Further, the substrate specificity of 19 kDa protein having a catalytic function is in good agreement with that of Oplophorus luciferase (Non-Patent Document 4).
Recently, it is reported that the mutated 19 kDa protein with the 16 amino acid mutations showed approximately 10-fold higher luminescence activity than that of native 19 kDa protein, and the mutated 19 kDa protein with different amino acid codons was named nanoKAZ or nanoLuc. This mutant with a signal peptide sequence for secretion was shown to be secreted from cells (Patent Document 2, Non-Patent Documents 5 and 6). In addition, the detailed studies on the 16 mutated amino acids were performed and the mutant (named eKAZ) with 3 amino acid mutaions (V44I, A54I and Y138I) showed an efficient substrate for coelenterazine. However, eKAZ was not secreted efficiently from cells (Non-Patent Document 7).
On the other hand, nanoKAZ and nanoLuc showed that native coelenterazine is not used for an efficient substrate. In contrast, bis-coelenterazine, 6h-f-coelenterazine, etc. which show highly hydrophobic properties, are used for efficient substrates (Non-Patent Document 6). As these coelenterazines with high hydrophobicity showed high permeability in cell membrane, and caused a high background level of luminescence, and cannot allow to be imaging for a long period of time. From these reasons, it has been desired to develop a secreted luciferase mutant which can use native coelenterazine more efficiently and has luminescence ability as same as that of nanoKAZ.